When a molecule is irradiated with photons of a particular frequency, the photons are scattered. The majority of the incident photons are elastically scattered without a change in frequency (Rayleigh scattering), whereas a small fraction of the incident photons (approximately 1 in every 108) interact with a vibrational mode of the irradiated molecule and are inelastically scattered. The inelastically scattered photons are shifted in frequency and have either a higher frequency (anti-Stokes) or a lower frequency (Stokes). By plotting the frequency of the inelastically scattered photons against their intensity, a unique Raman spectrum of the molecule is observed. The low sensitivity of conventional Raman spectroscopy, however, has limited its use for characterizing biological samples in which the target analyte(s) typically are present in small quantities.
When a Raman-active molecule is adsorbed on or in close proximity to, e.g., within about 50 Å of, a metal surface, the intensity of a Raman signal arising from the Raman-active molecule can be enhanced. This enhancement is referred to as the surface-enhanced Raman scattering (SERS) effect. The SERS effect was first reported in 1974 by Fleishman et al., who observed intense Raman scattering from pyridine adsorbed on a roughened silver electrode surface. See Fleishman et al., “Raman spectra of pyridine adsorbed at a silver electrode,” Chem. Phys. Lett., 26, 163 (1974); see also Jeanmaire, D. L., and Van Dyne, R. P., “Surface Raman spectroelectrochemistry. 1. Heterocyclic, aromatic, and aliphatic-amines absorbed on anodized silver electrode.” J. Electroanal. Chem., 84(1), 1-20 (1977); Albrecht, M. G., and Creighton, J. A., “Anomalously intense Raman spectra of pyridine at a silver electrode,” J. A. C. S., 99, 5215-5217 (1977). Since then, SERS has been observed for a number of different molecules adsorbed on the surface of metal surfaces. See, e.g., A. Campion, A. and Kambhampati, P., “Surface-enhanced Raman scattering,” Chem. Soc. Rev., 27, 241 (1998).
The magnitude of the SERS enhancement depends on a number of parameters, including the position and orientation of various bonds present in the adsorbed molecule with respect to the electromagnetic field at the metal surface. The mechanism by which SERS occurs is thought to result from a combination of (i) surface plasmon resonances in the metal that enhance the local intensity of the incident light; and (ii) formation and subsequent transitions of charge-transfer complexes between the metal surface and the Raman-active molecule.
The SERS effect can be observed with Raman-active molecules adsorbed on or in close proximity to metal colloidal particles, metal films on dielectric substrates, and metal particle arrays, including metal nanoparticles. For example, Kneipp et al. reported the detection of single molecules of a dye, cresyl violet, adsorbed on aggregated clusters of colloidal silver nanoparticles. See Kneipp, K. et al., “Single molecule detection using surface-enhanced Raman scattering (SERS), Phys. Rev. Lett., 78(9), 1667-1670 (1997). That same year, Nie and Emory observed the surfaced enhanced resonance Raman spectroscopy (SERRS) signal, wherein the resonance between the absorption energy of the Raman-active molecule and that of the nanoparticle yield an enhancement as large as about 1010 to about 1012, of a dye molecule adsorbed on a single silver nanoparticle, where the nanoparticles ranged from spherical to rod-like and had a dimension of about 100 nm. See Nie, S., and Emory, S. R., “Probing single molecules and single nanoparticles by surface-enhanced Raman scattering,” Science, 275, 1102-1106 (1997); Emory, S. R., and Nie, S., “Near-field surface-enhanced Raman spectroscopy on single silver nanoparticles,” Anal. Chem., 69, 2631 (1997).
Even with the enhanced signal due to the SERS or SERRS effect, the use of Raman spectroscopy can be limited in diagnostic assays and applications requiring a high sensitivity. Accordingly, there is a need in the art for SERS-active reporter molecules that give rise to an increased Raman signal when compared to SERS-active reporter molecules known in the art. The presently disclosed subject matter addresses, in whole or in part, these and other needs in the art.